Fig. 5: Mechanistic studies on ferroptosis induced by PAA4 and PAA5.
a Schematic pharmacological pathways of hypercarbon-centered gold(I) cluster. b Content of PAA4, PAA5 and PA1 in the whole EJ cells and cell-extracted mitochondria determined by ICP-MS (cAu(I) clusters = 1.5 μM, cPA1 = 6.0 μM, 4 h incubation). c Release of Au(I) ions monitored by confocal microscopy in the 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-yl-3-phenylpropiolate stained EJ cells, and ROS release monitored by confocal microscopy in the 2′,7′-dichlorofluorescin diacetate (DCFH-DA, 10.0 μM) stained EJ cells treated with PAA4, PAA5 (1.5 μM, 4 h incubation) and PA1 (6.0 μM, 4 h incubation) relative to the PBS blank trial. Scale bar: 20 mm. Excitation: 488 nm. d TrxR activity inhibition upon the treatment with PAA4, PAA5 (1.5 μM, 4 h incubation) and PA1 (6.0 μM, 4 h incubation) relative to the PBS blank trial. e Time-dependent intracellular ROS change in EJ cells treated by PAA4, PAA5 (1.5 μM, 4 h incubation), and PA1 (6.0 μM, 4 h incubation). The ROS concentration of EJ cells before treatment is referred as the control value. f Flow cytometry analysis of ROS level in EJ cells treated by 2.0 μM PAA4 with or without 2 h pretreatment of DFO (100 μM) and NAC (3.0 mM). ROS was labeled with DCFH-DA (10.0 μM). g Western blot analysis of pH2AX in EJ cells treated by PA1 (8.0 μM), PAA4 (4.0 μM) or PAA5 (4.0 μM) for 1, 3, or 6 h, respectively. (n = 2 independent experiments). h mRNA level analysis of CYBA and CYBB in EJ cells treated by PAA4 or PA1 for 6 h. i Confocal images with the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) assay of EJ cells incubated with PAA4, PAA5 (1.5 μM, 4 h incubation) and PA1 (6.0 μM, 4 h incubation), respectively. Excitation: 488 and 561 nm. Scale bar: 20 mm. Asterisks (*) denote the statistical significance: 0.001< ** P ≤ 0.01, 0.0001< *** P ≤ 0.001, **** P ≤ 0.0001, P values were performed with one-way ANOVA followed by post hoc Tukey’s test. Data were expressed as mean ± SD (n = 3 independent experiments examined over triplicates). Source data are provided as a Source Data file.
